(a) Field of the Invention
The present invention relates generally to methods of producing recombinant peptides by using of novel carrier proteins derived from the wild-type staphylococcus nuclease and its mutants.
(b) Description of Prior Art
Peptides constitute a group of biomolecules for which there is an increasing demand in many fields of biological, medical and pharmaceutical research. The increasing popularity of genome-scale protein studies or proteomics is further to increase the use of peptides for functional characterization and target validation. In addition to the use of peptides as drugs (Latham, P. W., Nature Biotech. 17, 755-757, 1999), peptides are also tools for investigating protein-protein interactions and as lead molecules for drug design. Indeed, the bound conformations of peptides in complex with target proteins are commonly used as templates for the discovery of small-molecule drugs (Mazitschek, R. et al., Mini. Rev. Med. Chem., 2; 491-506, 2002). There is also increasing evidence for the existence of peptide-like, or naturally unfolded, proteins which are encoded by the genomes and endowed with critical functional activities (Wright, P. et al., J. Mol. Biol., 293; 321-331, 1999; Uversky, V., Prot. Sci., 11; 739-756, 2002; Dunker, A. et al., Biochemistry, 41; 6573-6582, 2002).
Currently, chemical methods are used for the preparation of a variety of pharmaceutical peptides such as calcitonin, PTH, bivalirudin or other hirudin analogs and insulin. These purely chemical methods require the condensation of the corresponding amino acids or peptide fragments and very often suffer from cost disadvantages due to the use of elaborate purification methods and sometimes unnatural amino acids required. Given the increasing demand for peptides in pharmaceutical and biotechnology research, it is somewhat surprising that the main source of peptides still comes from synthetic techniques. Although solid-phase synthesis can produce good yields of peptides, the cost of synthetic peptides becomes unviable and/or prohibitively high when the desired peptide is greater than 30 residues. Moreover, uniform isotopic enrichment with 15N/13C or 2H for NMR studies is practically impossible for larger peptide fragments by solid-phase peptide synthesis.
For many years, it has been a common practice to use fusion proteins for the expression of small peptides. The commercially available carrier proteins are GST from Pharmacia, CBD from NEB, and some others from Novagen. Most fusion carriers have been selected to increase the solubility of the fusion constructs and the fusion carriers have been so large that the final yields of the expressed peptides are very low. The large sizes of the fusion carriers also complicate the purification steps of recombinant peptides. These findings indicate that production of recombinant peptides has been problematic for many (often unknown) reasons. In particular, the large size of the fusion carrier often limits the final yield of the target peptide. Quite often, secondary cleavage sites release undesirable peptides from the fusion carrier, which complicate the purification procedure. Sometimes, the fusion protein needs to be solubilized in a suitable buffer to facilitate peptide release by use of a specific protease. This is especially the case when there is at least one cleavage site (e.g. by CNBr) within the targeted peptide. Moreover, the production of peptides for preclinical and clinical evaluations often requires multi-gram quantities (Latham, P. W., Nature Biotech. 17, 755-757, 1999). To achieve the latter goal, high-yield expression of the fusion protein and simplified downstream processing steps need to be developed by the engineering of new carrier proteins.
Recombinant production of peptides has many advantages over chemical (solid-phase) synthesis including potentially higher yield, lower cost, easier scale-up and less environmental contamination. Although any polypeptide chain can be theoretically expressed in any microbial system, expression of peptides can sometimes be problematic in microbial hosts, such as Escherichia coli. The stability of the peptide expressed often results in a diminishingly low yield. In fact peptides expressed in a host cell can be degraded quickly by endogenous proteases and assimilated by the host cell. To overcome this problem, peptides can be expressed as fusion proteins with a suitable carrier protein. The fusion protein may in addition direct the peptides to specific subcellular compartments or inclusion bodies with the goal of achieving high yield of expression and avoiding protease degradation. The most significant carrier proteins used for the expression of peptides are listed as follows.
BPI
Better reported methods to produce human a atrial natriuretic peptide (U.S. Pat. No. 5,851,802 and WO00/55322). The inventor designed a series of recombinant expression vectors that encode peptide sequences derived from bactericidal/permeability-increase protein (BPI) as carrier proteins.
Carbonic Anhydrase
Partridge et al described methods to produce recombinant peptides by use of carbonic anhydrase as the carrier protein (WO96/16297). Three peptides including GRF (1-41), GLP1(7-34) and PTH(1-34) had been successfully prepared in this system. Wagner et al also developed a process for the recombinant preparation of a calcitonin fragment by using the same carrier protein and the use of the fragment in the preparation of full-length calcitonin and related analogs (U.S. Pat. No. 5,962,270, and WO97/29127).
α-lactalbumin
Cottingham et al invented a process to produce peptides as fusion proteins of α-lactalbumin in the milk of transgenic mammals (WO95/27782). The fusion partner acts to promote the secretion of the peptides and allows a single-step purification based on the specific affinity of α-lactalbumin to its antibodies. The peptide is released from the purified fusion protein by a simple cleavage step and purified from the liberated α-lactalbumin by repeating the same affinity purification method. This route provided a particular advantage of producing peptides that require specific post-translational modifications.
β-galactosidase
Shen used β-galactosidase as a carrier protein to express pro-insulin in inclusion bodies (Shen S., PNAS, 281; 4627-4631, 1984). The isolated inclusion bodies were solubilized with formic acid and cleaved with cyanogen bromide. Kempe et al used β-galactosidase as a carrier protein to express multiple repeats of the neuropeptide substance P in inclusion bodies of E. coli (Kempe T. et al., Gene, 39; 239-245, 1985). The peptide was released from the fusion protein by CNBr cleavage in a formic acid solution. Lennick et al also used this protein in a fusion system to express human α-atrial natriuretic peptide (Lennick M. et al., Gene, 61; 103-112, 1987). The target peptide was inserted as multiple repeats and the purified inclusion bodies were solubilized with urea followed by endoprotease cleavage. Schellenberger et al reported a process to express insoluble inclusion bodies of a fusion protein encoding a substance P peptide with β-galactosidase (Schellenberger et al., Int. J. Peptide protein Res., 41; 326-332, 1993). The isolated fusion protein was treated with chymotrypsin to separate the peptide from the carrier protein.
Chloramphenicol Acetyltransferase
Dykes et al reported a method to express human α atrial natriuretic peptide as a soluble intracellular fusion protein with chloramphenicol acetyltransferase in E. coli (Dykes C. et al., Eur, J. Biochem, 174; 411-416, 1988). The fusion protein was proteolytically cleaved or chemically cleaved with 2-(2-nitrophylphenylsulphenyl)-E-methyl-3′-bromoindolenine to release the peptide.
Glutathione-S-Transferase (GST)
Ray et al used glutathione-S-transferase (GST) to carry salmon calcitonin as a soluble intracellular fusion protein. The peptide was purified after the fusion protein was cleaved with cyanogen bromide. Hancock et al fused human neutrophil peptide 1 (HNP-1) or a hybrid cecropin/mellitin (CEME) peptide with GST and expressed the fusion proteins as inclusion bodies (WO94/04688, and Ray et al., Bio/Technology, 11; 64, 1993). Williamson et al used the GST expression system for the rapid and economic expression of recombinant neurotensin peptide (Williamson P. et al., Protein Exp. and Purif., 19; 271-275, 2000).
L-ribulokinase
Callaway et al reported a process to use L-ribulokinase as a carrier protein to express a cecropin peptide (U.S. Pat. No. 5,206,154, and Callaway et al., Antimicrob. Agents & Chemo, 37; 1614-1619, 1993). The fusion protein was expressed as inclusion bodies. The fusion protein was first isolated and then solubilized in formic acid prior to CNBr cleavage.
gp-55 Protein
Gramm et al used a bacteriophage T4-encoded gp-55 protein to fuse a human parathyroid hormone peptide (PTH) (Gramm H. et al., Bio/technology, 12;1017-1023, 1994). The fusion protein was expressed as inclusion bodies. The inclusion bodies were reacted with milder acid to hydrolyze an engineered Asp-Pro cleavage site.
Ketosteroid Isomerase
Kuliopulos et al reported the expression in insoluble E. coli inclusion bodies of a fusion protein encoding multiple repeats of a yeast α-mating peptide and a bacterial ketosteroid isomerase protein (Kuliopulos A. et al., J. Am. Chem. Soc., 116; 4599-4607, 1994). The isolated fusion protein was solubilized with guanidine hydrochloride prior to cyanogen bromide cleavage. Majerle et al (Majerle A. et al., J. Biomol. NMR, 18; 145-151, 2000) have demonstrated that isotope-labeled peptides could be prepared based on the peptide expression system first described by Kuliopulos et al. It was shown that recombinant peptide production had potentially many advantages over the solid-phase method of peptide synthesis, especially for isotope-labeled peptides of ˜10 residues in size.
Ubiquitin
Pilon et al described soluble intracellular expression in E. coli of a fusion protein encoding peptides fused to ubiquitin. The fusion protein was cleaved with a ubiquitin specific protease (UCH-L3) (Pilon A. et al., Biotechnol. Prog., 13; 374-379, 1997). Kohno et al also used ubiquitin to fuse mastoparan-X, a tetrdecapeptide known to activate GTP-binding regulatory proteins (Kohno T. et al., J. Biomol. NMR, 12; 109-121, 1998).
Bovine Prochymosin
Hauht et al reported the expression of a fusion protein encoding an antimicorbial peptide designated P2 and bovine prochymosin as insoluble inclusion bodies in E. coli (Hauht et al., Biotechnol. Bioengineer., 57; 55-61, 1998). The purified inclusion bodies were solubilized in formic acid and cleaved with cyanogen bromide.
GB1 Domain
Darrrinm et al used the GB1 domain as carrier protein to express the inhibitory region of Ctnl, clp (Darrrinm et al., Biochemistry, 41; 7267-7274, 2003). The fusion strategy takes advantage of the small size, stable fold and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis (Huth J et al, Protein Science, 6; 2359-2364, 1997). Pei et al used the GB1 domain as a solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins (Pei et al., J. Biomolecular NMR, 2001).
RNA-Binding Domain
Sharon et al reported an expression system to produce the 23-residue V3 peptide, the third variable loop of the envelop glycoprotein (gp120) of the HIV virus, linked to a derivative of the RNA-binding domain of the human hnRNP C protein (Sharon M. et al., Protein Exp. and Purif., 24; 374-383, 2002).
SH2 Domain
Fairlie et al reported the use of the N-terminal SH2 domain of the intracellular phosphatase, SHP2, as a carrier protein to express six peptides of ˜14 residues in length. This small protein domain confers an advantage for the production of disulfide-containing peptides (Fairlie W. et al., Protein Exp. and Purif., 26; 171-178, 2002).
A number of other publications have reported alternative peptide expression systems and described their utility for the production of one or two specific peptides (Baker, R., Curr. Opin. Biotechnol., 7; 541-546, 1996; Campbell, A. et al., Biochemistry, 36; 12791-12801, 1997; Jones, D. et al., Biochemistry, 39, 1870-188, 2000; Lindhout, D. et al., Biochemistry, 41; 7267-7274, 2002; Sprules T. et al., J. Biol. Chem., 278; 1053-1058, 2003). In general, the target peptides are fused to a highly expressed carrier protein in order to overcome the problem of low yields of peptide production. In some cases a carrier protein with low solubility has been exploited to direct the peptide to the inclusion bodies, thereby minimizing proteolysis and simplifying purification (Kuliopulos A. et al., J. Am. Chem. Soc., 116; 4599-4607; 1994; Majerle A. et al., J. Biomol. NMR, 18; 145-151, 2000; and Jones, D. et al., Biochemistry, 39; 1870-188, 2000).
However, there is still the question of expression yields and whether the available method is suitable for the production of peptides of larger sizes. Currently available methods for peptide expression also have many technical problems especially for the production of pharmaceutical peptides of small to medium sizes (e.g. >30 residues), which are often required for reducing side effects. Practically, it is very difficult to produce peptides using a normal recombinant system such as the GST fusion expression vector. The peptides are either not expressed or degraded by proteases for unknown reasons.
It would be highly desirable to be provided with a new fusion protein overcoming the drawback of the prior art, for the production of recombinant peptides.